Device for carrying out an almost simultaneous synthesis of a plurality of samples

ABSTRACT

The invention relates to a device for carrying out an almost simultaneous synthesis of a plurality of samples. The device is especially suitable for use in automated laboratory processes in the area of combinatorial chemistry. The aim of the invention is to provide a device of this type which enables the synthesis of a plurality of samples bonded to microbeads, said microbeads being provided in the cavities of a support plate. To this end, a plane support plate ( 1 ) is provided with a plurality of cavities ( 11 ) arranged regularly in an iterative grid. The cavities accommodate microbeads ( 12 ). A removable covering ( 2 ) is provided, said covering being provided with webs ( 21 ) which each cover at least one of a row of associated cavities ( 11 ) in such a way that a capillary gap ( 3 ) is formed between the microbeads ( 12 ) and the webs ( 21 ) and larger recesses ( 22 ) are left respectively between the adjacent webs ( 21 ). A dosed liquid dispenser ( 4 ) is allocated to each capillary gap ( 3 ).

BACKGROUND OF THE INVENTION

The invention relates to a device for a substantially simultaneoussynthesis of a plurality of samples that are particularly for use in theautomated laboratory work in the field of the combinatorial chemistry.

Sample particles, (“beads” or “Perlen”), have been used in separationsand synthesis in the laboratory technical field for tenth of years.These particles mostly are glass or polymeric globules that havediameters of 0.01 mm up to 1 mm, typically about 0.1 mm, which arefilled, dry or pre-swelled, as a loose material into a receptacle wherethey are then flushed by a liquid, whereby an adsorption process or areaction process takes place between the solid phase surface of theparticles and the liquid surrounding the particles. Methods of thecolumn chromatography (for example, gel filtration), of the columnextraction, of the immundiagnosis, of the bio-molecule purification (forexample, DNA cleaning), as well as of the homogeneous and heterogeneoussynthesis (of oligonucleotides, peptides or combinatorial substancelibraries) utilize these techniques. In addition to the automation andminiaturizing of laboratory techniques, the parallelizing of the same isof great interest in obtaining a higher sample throughput and, hence, tospeed up otherwise time-consuming procedures. To this end, samples arevery often arranged in a raster so that the identity (origin, quality)of the sample can be associated to an area coordinate. Such coordinatesare very easily to be detected in particular in automated systems ofsample handling.

Therefore, so-called micro-titer plates have been developed for liquidsamples, which support cavities in right-angular arrangements of 8·12(96 samples) 16-24 (384) or 32·48 (1536). Thereby, the dimensions of thecavities of these sample holders depend on such volumes that can bereliably dosed by the commercially available devices (pipettes), andfollow a miniaturizing continuously progressing with the dosingtechnology, what is simplified by the capability of an ali-quote(distribution of a mother-sample into different daughter-samples) ofliquids at will.

Within the frame of work for miniaturizing laboratory procedures thereis searched for possibilities to distribute sample particles, in analogyto the arrangement of liquid samples, in a two-dimensional raster. Sincethe miniaturizing of dosing liquids has already advanced very far, sothe single particle becomes the smallest unit. Furthermore, there is thedemand to handle high quantities, as it is common use when working withparticles. 1-g polymer resin contains about 1 million particles.

There are different solutions known for filling micro-titer plates orreaction vessels.

So WO 98/24543 A1 describes a device for transferring liquids in which,inter alia, a micro-titer plate is provided, the chambers of which haveat least one opening in their bottom area that is dimensioned in a waythat, in the course of a filling operation, the passage of the liquidthrough this opening due to capillary forces is avoided. In WO 98/06490A1 a device for an organic solid phase synthesis is described, in whichthe reaction vessel is arranged above a collective vessel for receivingliquids in such a way that the transfer of the liquid can be attained bygenerating a low pressure, whereas the liquid is held in the reactionvessel by a slight overpressure. From WO 97/19749 A1 there is a devicefor addressable combinatorial substance libraries known, in which onesubstance each is represented in a capillary tube, whereby the fillingwith liquids is obtained by capillary forces. In WO 97/37755 A1 a plateis described that contains a plurality of reaction cells being arrangedin rows and columns, which are supplied with liquids by pumps.Furthermore, the specifications WO 97/43629 A1 and WO 98/16315 A1describe distribution systems for liquids, which are comprised of aplurality of plates, whereby the liquid flow is operated and controlledby a capillary barrier and by electro-kinetic pumps, respectively.

The miniaturizing of the support plates mentioned goes along with theminiaturizing of the corresponding filling technologies and meets itscritical geometric or time limits when conventional automated pipettingdevices are used, since each single sample particle has to be suppliedwith liquids.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a device for asubstantially simultaneous synthesis of a plurality of samples, whichare bound to micro-beads provided in the cavities of a support plate.

The object is realized by the features of the first patent claim.Advantageous embodiments are covered by the dependent claims.

DETAILED DESCRIPTION OF THE INVENTION

In the following, the invention will be explained in more detail byvirtue of schematical embodiments. There is shown in:

FIG. 1 a perspective view of a principle setup of an inventional deviceas well as a representation of an enlarges detail;

FIG. 2 a a lateral view of a device according to FIG. 1;

FIG. 2 b a plan view of a device according to FIG. 1;

FIG. 3 a plan view of a device according to FIG. 1 with the position ofan inventional cover in two synthesis steps; and

FIG. 4 a plan view of an embodiment of a cover with a plurality ofoperational sections.

Without limiting the invention thereto, it will be started in FIG. 1from a support plate 1, in which micro-beads 12 are provided in each ofthe single cavities 11 in such a way that the micro-beads project fromout of these cavities. When micro-beads of a diameter of 100 μm areused, these micro-beads in the sorted-in state will project from out ofthe surface of the support plate 1 by of from 20 to 50 μm. In thepresent example, each nine of such micro-beads belong to one samplereceiving area, whereby all the sample receiving areas are aligned toone another in rows and columns (refer to FIG. 3). A detachable cover 2is provided above the bead-filled support plate 1, the cover 2 supportsbarriers 21 as most substantial elements. These barriers are so designedin their width and length that they capture all sample-receiving rangesof one row or column when attached to the support plate 1. In thismanner, due to the definedly preselectable projecting of the micro-beads12, a capillary gap 3 of preselectable height and of a defined width isproduced, the latter by preselection of the width of the barriers 21. Ina further arrangement of the micro-beads, for example, a plurality ofset back micro-beads in a common cavity, such a capillary gap can bealso formed in that the support plate 1 is provided with spacers of adefined height in the range, where the barriers 21 are supported, and/orthe barriers 21 themselves are provided with spacers of a definedheight.

In order to determine the sides of the capillary gaps 3, which areformed in this manner, the barriers 21 are spaced from one another bylarger recesses 22, which are dimensioned in such a way that capillaryforces will not be active any longer in these recesses.

The height of the capillary gap 3, at which the capillary forces willstill be active in transporting liquids, depends on the surface tensionof the material for the support plate 1 and for the barriers 21, and onthe liquids that are to be handled. When materials such as glass andmetal are used, between which a gap is formed, and water is used as aliquid, the capillary forces will be active up to gap heights of 500 μm.When using water, a specific hydrophilizing of the glass and/or of themetal surface permits the flowing of a liquid due to capillary forcesalso at greater distances. Depending on the synthesis fluid that is tobe handled, the barriers 21 in the range of their supporting area on thesupport plate can be provided with a hydrophilic or hydrophobic surface,whereby at least the side walls of the recesses 22, which limit thebarriers, should be provided with a respective oppositely acting surfacecovering.

The capillary gap, formed in the example by the projecting-outmicro-beads 12, opens up the possibility to have liquids intentionallyflow along the gap. Thereby a structurized cover plate is used for thecover 2 in the example, whereby this cover plate is provided withparallel recesses 22. By these recesses 22 a separation of two capillarygaps running in parallel to one another is obtained.

The capillary gaps will be filled in that a liquid is pipetted at theleading face of the cover, into a respective start of the gap, as isschematically indicated by way of a gap and by means of a dosable fluiddispenser 4 in FIG. 1.

The filling of each capillary gap with a different liquid requires avery careful pipetting of the liquids in order to prevent the mixing oftwo liquids. In order to ensure a simultaneous and equally dosed fillingof all capillary gaps 3, it is more advantageous to provide the liquidsupply via bores 13 in the support plate 1 or via bores 25 in the cover2, which are respectively pre-positioned to a row and column of thecavities arranged in a line. A connection is provided via hose-likeconnections 5 or fitting pieces connected to these bores, to a liquidsupply means, for example, a jet-pump, not shown in more detail in thefollowing. This liquid supply means is associated to a respective row orcolumn, whereby it is advantageous to apply a common and equally definedpressure to all liquid supplies. Such connections are adapted to feednow well-dosed liquids into the capillary gaps. It is, however,necessary to very precisely adapt the pumping rate of the jet-pump tothe speed of flow of the liquid, produced by the capillary forces. Thishas to be detected by experiment for the individual case, in order toavoid a crossover of the liquid from a capillary gap to an adjacent one.

As already hinted at, it is also possible to realize the filling of thecapillary gaps via the cover plate 2. Then the bores for the connectionsof the hoses have to be produced in the cover plate, centrally to thebarriers 21. Such an embodiment also permits to use an adapter for amicro-titer plate instead of the hose connection, and to realize thefilling of the capillary gaps by exploiting hydrostatic differences inpressure. One advantage in using jet-pumps connected to hose connectionsis the compactness of the system so that evaporations are avoided. Sucha closed system is also advantageous in those cases where chemicals areused which must not get into contact with air.

The described modifications permit the filling of any desired number oflines with liquids. Presently, in adaptation to the micro-titer platesavailable, the described device permits the simultaneous filling of 96lines. There are, however, no limits as to the number of lines, and withan increasing use of micro-technical machining processes there can berealized far more than 100 lines.

FIG. 2 a shows an embodiment of the device according to FIG. 1 in alateral front view, thereby the support plate 1 is provided with bores13, only five of which are represented in FIG. 2 a. Hose-likeconnections 5 are connected to these bores 13, said connections lead tonot shown liquid supply means 4. The support plate 1, in turn, ismounted on a displacement table V, which enables a lateral displacementin parallel to the normal of the sheet. Furthermore, the support plate 1and the cover 2 are connected to each other via a guiding means 6. Sucha design permits to incorporate a further structurized cover 2, as it isshown in plan view in FIG. 2 b in more detail. In addition to thetransparent cover 2, described hereinbefore, including the barriers 21provided to the same, the cover 2 further comprises a porous portion 23,which is followed by a plane section 24. The extensions of this planesection 24 are dimensioned in such a way that the plane section iscapable of covering the entire support plate 1 under formation of acapillary gap capturing all the sample areas, provided that the coveringrange has been moved over the support plate 1. A complete design of onlysuch a coverage alone is shown in FIG. 4, in which the bores 25, as analternative, are there allocated to the first part of the cover 2.

Under use of the device described, the actual synthesis of the desiredsamples is performed as follows: The support plate 1, having a size of250·250 mm2, which is filled with micro-beads 12 that, at a suitableporosity, can take, for example, a sample liquid volume of 0.25 nl, isbrought into contact with the cover 2 containing the recesses 22. Thesupport plate 1 and the cover 2 are aligned to each other by means of anadjustment device, not shown in detail, so that one projecting barrier21 each rests upon a row of reaction chambers filled with beads. Thebarriers 21: connect, for example, one row with 96 sample fields thatcomprise 864 micro-beads 12. Moreover, the support plate in the examplehas two-times 96 through-bores 13 that lie in the extension of the rowsand columns of the arrays of beads. The above-described hoses 5 aresecured to these bores on the rear side of the support plate. The 192hoses lead to the liquid supply means, for example syringes, which is,respectively, are filled with chemicals. A pressure is simultaneouslyapplied to the syringes by a syringe drive, and the liquids aretransported to the support plate 1 via the hoses 5. The capillary gaps 3are filled with one chemical each. This is achieved in that the end ofeach barrier is placed accurately above one bore, from out of which theliquid is ejected for entering into the capillary gap. The pumping rate,which is controlled by the syringe drive, has to be correlated to thespeed of flow of the liquids, driven by the capillary forces. A fillingoperation will take about 3 min., when a capillary gap of a height ofabout 30 μm, a width of about 1000 μm and a length of 250 mm is used.

The support plate 1 is mounted upon the displacement table V, whichpermits movements in parallel to the barriers 21, whereas the cover inthe example should be safely arrested during the entire synthesis.Following the first synthesis step, the support plate I will bedisplaced underneath the cover 2 and along the barriers 21, in thecourse of which the micro-beads are conducted past by the porous portion23 of the cover 2. Thereby the size of the pores in the porous portionhas to be significantly smaller than the diameter of the micro-beads.The porous region absorbs the synthesis chemicals and these chemicalsare drained from there by means of a device operating with low pressureand which is not represented in more detail; the micro-beads 12 aredried in this way. The support plate 1 is completely moved past belowthe porous area, until the entire support plate has arrived at the backend portion of the cover that is given a plane surface. In this section24 the support plate 1 is subject to a rotation of 90°, which isnecessary for performing the second synthesis step. Also in this casethe support plate 1 is moved by means of a rotary table, while the coverremains safely clamped. After the support plate 1 has been rotated, themicro-synthesis beads are subject to a flushing. The flushing-outsolution will also be transported by capillary forces to the beads.Again a drying of the beads is carried out by wiping over the porousportion 23 of the cover as described hereinabove. Then the cover 2 isagain in a position for synthesis. The second synthesis step nowproceeds in a same manner, the support plate 1, however, lies under thecover plate rotated by 90° and a further coupling step follows. Thereare further coupling steps possible, as many as desired. Thus, after thetwo-stage synthesis described, and starting from 96 rows and 96 columns,9216 different combinations of twice 96 substances result. Furthersynthesis steps, after a respective rotation of the plates relative toeach other, are feasible in any desired number. Two of the synthesispositions described are exemplified in FIG. 3 by example of sixteen rows(1 to 16) and sixteen columns (A to Q).

The use of borofloat glass for the cover 2 has proven as beingparticularly advantageous. In order to be able to visually supervise theliquid stream, which forms in the capillary channels mentioned, atransparent material should always be selected for the cover 2.Additionally, glass is distinguished by a high degree of flatness, whichis is an important criterion for realizing a capillary gap of uniformthickness extending over a length of 250 mm. By use of diamnond cuttingtools, 97 recesses 22 of a distance of 2.25 mm have been worked into afirst range of the cover, under the condition that there are 96 rows andcolumns, respectively. The depth and width of a recess is so dimensionedthat the recess 22 itself does not act like a capillary any more. Tothis end a width of 1000 μm and a depth of 1500 μm are selected in theembodiment described, thus barriers 21 of a width of 1.25 mm remainbetween two adjacent recesses. It lies within the scope of the inventionto use other materials for the cover.

The design of the cover 2 described hereinabove is the most advantageousone, as concerns handling and stability of the device. It, however, alsolies within the scope of the invention to realize the provided barriersby use of an arrangement of parallel stripes. To this end single stripesof glass are used correspondingly dimensioned in length and width. Theheight of the stripes can be selected as desired, and only depends onthe stability requested from the device. The single stripes will bearranged parallel to each other at the distance of the sample receivingranges, and they will be fixed relative to each other by sticking theirends onto a supporting stripe or to a support plate.

List of Reference Numerals

-   1—support plate-   11—cavities-   12—micro-beads-   13,25—bores-   2—cover-   21—barriers-   22—recesses (between the barriers 21)-   23—porous portion-   24—plane section-   3—capillary gap-   4—liquid supply means-   5—connections (hoses)-   6—guiding means

1. Device for a substantially simultaneous synthesis of a plurality ofsamples comprising a support plat having a plurality of cavities which,under formation of rows, are regularly arranged in a repeated raster,and which are adapted to receive micro-beads, wherein a detachable coveris provided, which has barriers of a defined width, said barriers coverand space apart at least one respective cavity associated to one row ofcavities in such a manner that capillary gaps are formed between themicro-beads and the barriers, wherein the capillary gaps are formed by aspace resulting from the micro-beads projecting from out of thecavities, with one dosageable liquid supply means being associated toeach of said capillary gaps, and so large a recess remains between tworespective adjacent barriers that said recess is capillary inactive. 2.Device as claimed in claim 1, wherein the cover is formed by atransparent plate, into which parallel indentations are inserted forformation of larger recesses.
 3. Device as claimed in claim 1, whereinthe barriers are provided with a hydrophilic or hydrophobic surface inthe area where contacting the support plate, whereby at least the sidewalls of the recesses limiting the barriers are provided with arespective oppositely active surface coat.
 4. Device as claimed in claim1, wherein the support plate and the cover are mounted, by means of aguide, relative to each other in a connection which is laterallydisplaceable and rotatable by 90°.
 5. Device as claimed in claim 4,wherein, subsequent to the barriers at least one porous portion and afurther plane section are allocated to said cover, whereby said afurther plane section covers the entire support plate.
 6. Device asclaimed in claim 1, wherein the liquid supply for the gaps takes placein the support plate via bores, each respective bore beingpre-positioned to a respective row of cavities.
 7. Device as claimed inclaim 1, wherein the liquid supply for the gaps takes place in the covervia bores, each respective bore being pre-positioned to a respective rowof cavities.
 8. Device as claimed in claim 6 or 7, wherein said boresare provided with hose-like connections or fitting pieces, which areeach connected to a liquid supply means, a defined pressure beingadapted to be applied to said liquid supply means.